The invention relates to protein-binding media for affinity chromatography and especially to solid media having high specific protein-binding capacity.
Affinity chromatography involves separation of proteins by selective absorption onto and/or elution from a solid medium, generally in the form of a column. The solid medium is generally an inert carrier matrix to which is attached a ligand having the capacity to bind under certain conditions the required protein or proteins in preference to others present in the same sample, although in some cases the matrix itself may have such selective binding capacity. The ligand may be biologically complementary to the protein to be separated, for example antigen and antibody, or may be any biologically unrelated molecule which by virtue of the nature and steric relationship of its active groups has the power to bind the proteins.
There are described in, for example, U.S. Pat. No. 4,016,149 and by Baird et al, Febs Letters, Vol 70 (1976) page 61, solid media wherein the ligands are mono-chloro-triazinyl dyes which are bound to dextran or agarose matrices by substitution of the chloride substituent. Such binding is carried out in sodium carbonate or bicarbonate buffered media and since the dyes are designed for dying cellulose, the bound dye concentrations on non-cellulosic matrices are generally very low resulting in low protein binding capacity. It is possible to increase the dye binding by cyanogen bromide activation of the agarose matrix. However cyanogen bromide activation has serious disadvantages, especially for industrial use: the material is highly toxic and hence too dangerous to use on an industrial scale; it produces amido carbonate and carbamate links with the matrix which are unstable and cause gradual loss of column activity in long-term use; and it activates hydroxyl and amine groups on the dye resulting in dye-dye bonding and dye-bonding other than through the triazine group so that only a small proportion of the bound dye molecules are available for protein binding and their protein binding properties may differ. In addition, it is sometimes found that columns with very high bound dye contents bind protein so firmly that elution is not practicable. Moreover cyanogen bromide activation cannot be used for immobilisation of dichloro-triazinyl dyes.
A further class of solid media are prepared by linking monochloro-triazinyl dyes to dextran and then to agarose supports. In this case the chlorine substituent on the triazine ring is replaced by an -O-dextran linkage and linkage to the support is entirely via other reactive, especially amine, groups on the dye following cyanogen bromide activation. Hence the binding specificity again differs from media wherein the binding is via the triazine ring, and the media suffer all the disadvantages discussed above in respect of cyanogen bromide activation.
There is thus a need for a method of achieving useful controlled levels of dye binding without the use of cyanogen bromide.
The present invention provides a process for producing a protein binding solid medium, said process comprising reacting a protein binding ligand material of structure ##STR1## wherein R.sup.1 is a sulphonated derivative of anthraquinone, a substituted anthraquinone, an aromatic azo group or a phthalocyanine group, and R.sup.2 is either an organic group or a chloro substituent, with an affinity chromatographic matrix containing hydroxy or amino groups which is a polymer or copolymer of agarose, dextrose, dextran or acrylamide, at a pH of at least 8, in an aqueous solution of an alkali metal hydroxide and an alkali metal salt wherein the salt enhances the binding of the ligand material to the matrix by a common ion effect.
In the case of monochlorotriazinyl ligands (R.sup.2 is an organic group) useful levels of dye binding may also be achieved in the absence of the alkali metal salt.
It has surprisingly been found that the use of an alkali metal hydroxide in this reaction, particularly if the use is in conjunction with a suitable alkali metal salt, results in a much higher level of binding of the ligand to the matrix than occurs if the reaction is conducted under similar conditions of temperature, time and base concentration but in the presence of an alkali metal carbonate or bicarbonate. Furthermore, the media produced by the present process show tighter elution profiles than their carbonate/bicarbonate produced counterparts.
The alkali metal hydroxide may be any Group I metal hydroxide. Generally however the more readily available hydroxide bases such as potassium and, which is particularly preferred, sodium hydroxide will be employed.
The alkali metal salt may be any Group I metal salt which, in conjunction with the chosen alkali metal hydroxide, enhances the binding of the ligand material to the matrix by a common ion effect. Examples include potassium chloride, sodium sulphate, di-sodium hydrogen phosphate, sodium nitrate and, which is particularly preferred, sodium chloride.
In a preferred embodiment of the present process the ligand material and the matrix are first mixed in a suitable solvent in the presence of the alkali metal salt. The alkali metal hydroxide is then added and the binding reaction allowed to proceed.
The ligand material is a triazinyl dye, especially those sold under the Trade Marks "Cibacron" and "Procion". In one preferred embodiment of the present invention R.sup.1 is a sulphonated derivative of anthraquinone or a substituted anthraquinone. In this case R.sup.1 may be a derivative of structure ##STR2## wherein R.sup.3, R.sup.4 and R.sup.5 each represent a sulphonyl group or a hydrogen atom. Optionally such a derivative may be further substituted by alkyl or amino groups.
In an alternative preferred embodiment R.sup.1 is a sulphonated derivative of an aromatic azo group. In this case R.sup.1 may be a derivative of structure ##STR3## wherein R.sup.6, R.sup.7 and R.sup.8 each represent a sulphonyl group or a hydrogen atom and the point of attachment to the triazinyl ring may be any of the points marked "*", Optionally such a derivative may be further substituted by other suitable substituent groups.
R.sup.2 may be either an organic group or a chloro substituent. In the former case R.sup.2 is preferably a sulphonated aromatic, particularly a sulphonated phenyl, group.
Examples of triazinyl dyes that may advantageously be employed in the process of the present invention are the Cibacron dyes manufactured by Ciba Ltd and the Procion dyes manufactured by ICI. Illustrative of these dyes are
Cibacron Orange G-E, Cibacron Brillant Blue FBR-P, PA0 Cibacron Blue F3G-A, Cibacron Brillant Red 3B-A, PA0 Cibacron Brown 3 GR-A, Cibacron Scarlet 2G, Cibacron Scarlet 4G-P, PA0 Cibacron Brillant Red B-A, Procion Brillant Orange HGRS, PA0 Procion Blue HBS, Procion Brillant Red H7BS, Procion Orange PA0 Brown HGS, Procion Scarlet H3GS, Procion Red H3B, Procion Red HE3B, PA0 Procion Red P3BN, Procion Red MX2B, Procion Blue MX3G, PA0 Procion Yellow MXR, Procion Yellow H5G, Procion Red H8BN, PA0 Procion Green H-4G, Procion Brown MX5BR, Procion Blue MX-G, PA0 Procion Blue HE-RD, Procion Blue H-B, Procion Blue MXR, PA0 Procion Yellow HA and Procion Green HE-4BD.
When commercial dyes are used it may be necessary to remove wetting agents by, for example, washing with ether or acetone.
The matrix is a polymer or copolymer of agarose, dextrose, dextran or acrylamide, with an agarose homopolymer being particularly preferred. (NB. Cellulose and substituted celluloses are unsuitable as matrices since although they bind large quantities of dye, this is poorly accessible to protein during chromatography resulting in poor protein binding.)
The optimum concentration of alkali metal hydroxide depends on the structure of the ligand. For monochloro-triazinyl derivatives (R.sup.2 =an organic group) the pH should be at least 9.5 to achieve an optimum coupling. Normally the alkali concentration should be 0.02 to 0.4, preferably 0.05 to 0.2N, although the upper limit is not particularly critical.
With dichloro-triazinyl derivatives (R.sup.2 =chlorine) the alkali concentration should be about 0.002 to 0.1, preferably 0.005 to 0.01N (pH about 8 to 12.5) and the ligand binding is found to fall off quite rapidly once an optimum alkali concentration is exceeded.
The ligand-matrix coupling reaction may be conducted over a wide range of temperature, within the stability ranges of both reagents, without serious effect on the amount of ligand bound. However the mono-chloro triazinyl ligands bind only slowly so that at ambient temperatures (15.degree.-25.degree. C.) as long as 40 to 60 hours may be required for optimum reaction, and elevated temperatures of 40.degree. to 60.degree. C. are preferred since they both speed the reaction and yield a media showing a tighter elution profiles. Dichloro-triazinyl-ligands normally react in 1 to 4 hours of ambient temperatures and there appears no significant advantage in using higher temperatures.
The concentration of the alkali metal salt should be high enough to produce an enhancement but not so high as to reverse the effect. Typically the alkali metal salt concentration will be between 0.25 and 0.5 Molar. Such a concentration of sodium chloride at least doubles the amount of dye bound to the matrix (in comparison with the amount of dye bound in the absence as NaCl).
The presence of chloride substituents in the solid medium may have an adverse effect on protein-binding and hence when dichloro-ligands are used in the above process, the process should preferably be followed by a further step to convert any free chloride substituent in the solid medium to a less displaceable group such as amino, hydroxyl or thiol.
The process of the present invention permits much higher concentrations of dye to be bound to the matrix than was possible with previously known non cyanogen bromide based methods. Thus according to a further aspect of the invention there are provided protein-binding solid media comprising a protein-binding ligand containing triazinyl groups bound directly to a matrix and having substantially the structure. ##STR4## wherein X represents --O-- or --NH-- and R.sup.1 and R.sup.2 are as defined above, said ligand being present in a proportion of more than 15 mg preferably at least 30 mg especially at least 45 mg per gm of dry matrix. These figures are for the ligand actually bound as determined by digestion in 50% (v/v) acetic acid followed by spectrophotometry. It should be noted that although these amounts are less than those sometimes quoted in the prior art, the prior art figures refer to the amount of dye added to the reaction mixture not to the amount of dye actually bound to the matrix.
Preferably the matrix is an agarose homopolymer.
In accordance with yet another aspect of the invention, the protein-binding solid media may be used in the conventional manner for the separation and purification of proteins by affinity chromatography. Thus a crude or semi-purified biological material normally in a buffered solution at a concentration of 1 to 20, but exceptionally up to 100, mg protein per ml, is contacted, normally in a column, with the solid medium. After washing the medium the retained protein may be eluted by standard techniques such as by using a solution of different pH, buffer composition or ionic strength, or containing a co-substrate, co-factor, inhibitor, allosteric effector, unbound ligand or chaotrophic reagent or by electrophoresis.
The media of the present invention are capable of performing simple and highly specific purifications of a wide range of proteins, especially blood proteins and enzymes, both animal and bacterial. For example, selected media may be used to purify albumin, kinases such as glycerokinase and especially urokinase, nucleases including restriction endonucleases, dehydrogenases such as glyceraldehyde-3-phosphate dehydrogenase or .beta.-hydroxy-butyrate dehydrogenase, esterases such as cholinesterase, or DNA or RNA binding proteins such as DNA lygase. Because of the high bound-ligand content of the media, the amount of protein which may be separated per gram of medium is very much higher contents may result in excessive binding of the protein and consequent elution difficulties, an effect not encountered at ligand concentrations achieved by the prior art. Thus in these cases optimisation of the ligand content is necessary.
The various aspects of the invention will now be illustrated by reference to specific examples of the preparation and use of protein-binding solid media, wherein all bound-ligand figures are quoted on the basis of weight of media sucked dry on a filter funnel. Thus 15 grm sucked dry medium equals about 25 ml settled volume and 1 grm of fully dried material. It should be noted that the degree of ligand binding varies between dye batches and with the age of the dye so that results of experiments run at different times are not always strictly comparable.
The ligands used in these examples were commercial dyes having the following typical structures and Colour Index Constitution Numbers (CICN). In all cases M may represent a hydrogen atom, but normally represents an alkali metal, usually sodium, ion. All dyes were ether washed before use.
TABLE 1 __________________________________________________________________________ LIGAND CICN No TRADE NAME STRUCTURAL FORMULA __________________________________________________________________________ A (n = 1) 18159 Procion Red H3B ##STR5## B -- Procion Red HE3B As Ligand A, but n = 2 (p-isomer) C -- Procion Red P3BN Similar to Ligand A D 18158 Procion Red MX2B ##STR6## E -- Procion Blue MX3G ##STR7## F 13190 Procion Yellow MXR ##STR8## G 18972 Procion Yellow H5G ##STR9## H 61211 Cibacron Blue F3GA ##STR10## I -- Procion Red H8BN ##STR11## __________________________________________________________________________ Procion and Cibacron are Trade Marks